a novel rice ERF gene, up-regulates ethylene-responsive genes ...

1718 et al., 2004;

1718 et al., 2004; Nakano et al., 2006). They are characterized by the presence of the highly conserved AP2/EREBP DNA-binding domain of about 60 amino acids (Weigel, 1995; Okamura et al., 1997). Depending on the number of domains, the superfamily is divided into AP2 and EREBP subfamilies. Members of the AP2 subfamily have two domains in tandem while members of EREBP subfamily contain only one (Weigel, 1995; Okamura et al., 1997; Sakuma et al., 2002). Notably, all of the characterized AP2 subfamily members are found to be development-related (Nakano et al., 2006). In contrast, EREBPs play roles in response to phytohormone, pathogen attack and environmental stresses such as cold, drought and high salt (Liu et al., 1998; Haake et al., 2002; Dubouzet et al., 2003; Gutterson and Reuber, 2004). Based on their function and conserved amino acids of their AP2/EREBP domain, presently known EREBPs can be further assigned into the ethyleneresponsive factor (ERF) or dehydration-responsive element-binding protein (DREB) subgroup (Sakuma et al., 2002). Amino acids 15 and 20 in the domain of DREBs are V (Val) and E (Glu), respectively, in contrast to A (Ala) and D (Asp) in ERFs. This divergence is supposed to explain the functional differences between the two subgroups (Sakuma et al., 2002): DREBs such as OsDREB from Oryza sativa and DREB1 from Arabidopsis are implicated in responses to abiotic stresses (Liu et al., 1998; Dubouzet et al., 2003; Guo et al., 2004), and ERFs function mainly in biotic stress-resistant responses (Gutterson and Reuber, 2004), examples being ERF1, ERF2, ERF3 and ERF4 from Arabidopsis thaliana (Zhou et al., 1997; Solano et al., 1998; Gu et al., 2000; Berrocal-Lobo et al., 2002) and Pti from Solanum tuberosum (Gu et al., 2000, 2002). In rice, more than 100 genes have been predicted to encode ERF proteins (Nakano et al., 2006). However, functional information are currently available only for a few genes such as FZP and BIERFs: FZP is identified to play a crucial role in establishing floral meristem identity and BIERFs show inducible expression to 2,1,3-benzothiadiazole, a plant systematic resistance activator (Cao et al., 2006; Nakano et al., 2006). Further investigations of functional characteristics of more ERFs are essential to extend our understanding of their roles in plants. In Arabidopsis, several ERFs such as AtERF1, AtERF3 and ERF1 are found to be ethyleneresponsive (Solano et al., 1998; Fujimoto et al., 2000), but corresponding ricegenes involved in ethylene response have not been reported previously. In this study, we cloned a putative riceERFgene, OsERF1, and characterized its function by overexpression of it in Arabidopsis since network of ARTICLE IN PRESS gene regulation in Arabidopsis is far clearer than in any other plant species. Materials and methods Plant materials and nuclear acid extraction Rice plants (O. sativa L. ssp. japonica cv. Zhonghua 10) were grown under standard conditions. Leaves were collected from 2-week-old seedlings, and inflorescences were harvested at different stages. Young roots and buds were gathered from seeds germinated on sterile-water soaked filter papers for 3 d. To detect responses of the gene to plant growth substances or stresses, 2-week-old rice seedlings were transferred into solutions containing 0.1 mM ethrel (purity, 95%), 0.01 mM abscisic acid (ABA) (Invitrogen), 20% polyethylene glycol (PEG) ( 4.5 MPa), 1 mM salicylic acid (SA) and 0.1 mM methyl jasmonate (MeJA) with roots immersed completely in these solutions and cultivated at 28 1C for 4 h. For cold treatment, seedlings were exposed to 8 1C with a control at 28 1C for 4 h. To detect detailed responses of OsERF1 to ethrel inducement, 1 mM ethrel was employed to treat rice seedlings for 1, 2 and 4 h. Total RNA was isolated by the use of Trizol kit (Invitrogen) according to the manufacturer’s protocol and digested by RNase-free DNase (Takara) to remove residual genomic DNA. Cloning of full-length OsERF1 cDNA The full-length cDNA of OsERF1 was cloned by use of a RACE approach (Invitrogen 5 0 RACE kit), with the genespecific primers synthesized according to the predicted genomic sequence of OsERF1. Primers 5 0 -ATGCGGAGCGG- GAAGTTGA-3 0 and 5 0 -AATGTCGACTCCTCCTTCCCGTGC-3 0 were used for 5 0 RACE; 5 0 -GAGGTCGGGTCAGCAAGGAA-3 0 and 5 0 -TCCTCAACTTCCCGCTCCGC-3 0 for 3 0 RACE. RT-PCR analysis of OsERF1 mRNA accumulation An amount of 5 mg of total RNA extracted from a given tissue was reverse-transcribed into first-strand cDNAs by ReverTra Ace (Toyobo). RT-PCR was performed in 50 mL mixture including 5 mL of first-strand cDNA, 20 pmol of each of the gene-specific primers (5 0 -GGTGCAGGCATGG- TACCCC-3 0 and 5 0 -CCCTCACAAACTCACTCGG-3 0 ), 0.4 mM dNTPs, 1 GC buffer (Takara) and 2.5 U Taq DNA polymerase (5 U/mL, Takara) for 35 cycles. Rice Tubulin A cDNA (Tub A) was amplified for 25 cycles as a constitutive control (Ding et al., 2002). Construction of OsERF1 expression vector Y. Hu et al. A fragment spanning OsERF1 ORF was PCR-amplified from genomic DNA by use of the primers 5 0 -TTTCCATGGC- GATGACGGCGCGAAGCATG-3 0 and 5 0 -ACTACTAGTGATGAC- GAGCTGCTCCACGC-3 0 , which contained added NcoI and

SpeI enzyme sites, respectively. After digestion, the amplicon was inserted into the tool vector pCAMBIA1302 (CAMBIA) to produce OsERF1 expression construct pOsERF1, which contained an OsERF1:green fluorescent protein (GFP) fusion under the control of CaMV35S promoter. The resulting OsERF1 expression construct pOsERF1 and pCAMBIA1302 vector (used as a control) were first introduced into Agrobacterium tumefaciens strain EHA105 and then into A. thaliana var. Columbia by a floral dip method (http://www.nlh.no/research/narc/ protocols/floral_dip.htm). Subcellular localization of OsERF1 protein pOsERF1 was introduced into Allium cepa (onion) epidermis cells by A. tumefaciens transformation (Yang et al., 2000). GFP fluorescence signals were detected under microscopy with a fluorescein isothiocyanate (FITC) filter (Zeiss) after incubation of the transformed cells on the Murashige and Skoog (MS) (1962) medium at 25 1C in the light for 2.5 d. Subcellular localization of OsERF1 was also examined by use of the T3 seedlings of transgenic Arabidopsis line L6 expressing OsERF1:GFP. Screening of transgenic Arabidopsis seedlings Transgenic Arabidopsis seeds were germinated and grown on a solid MS medium supplemented with 25 mg/L hygromycin (hyg) in the dark for 3 d. Then, hyg-resistant seedlings were transferred to flower pots and maintained under standard conditions. Examination of insertion and expression of OsERF1 in transgenic Arabidopsis Genomic DNAs extracted from Arabidopsis wild-type and transgenic plants were digested completely with HindIII. About 20 mg of the digested DNAs were separated on a 0.8% agarose gel followed by denaturation and neutralization before being blotted onto a Hybond N + nylon membrane (Amersham Biosciences). After ultraviolet (UV) cross-linking, hybridization was carried out overnight at 65 1C with an [alpha- 32 P]CTP-labeled GFP fragment (spanning nucleotides 67–689 of the pCAM- BIA1302), which was labeled by use of the primer-a-gene labeling system (Promega). For RNA gel blot, 25 mg of total RNAs from transgenic and wild-type plants were fractioned on a 1.2% agarose–formaldehyde gel and transferred onto Hybond-N+ nylon membranes, followed by UV cross-linking. Hybridization was performed with the same probe as described for the DNA gel blot, and the resulting membrane was autographed at 80 1C. mRNA examination of ethylene-responsivegenes expression in transgenic Arabidopsis Semi-quantitative RT-PCR was performed to assess transcript accumulation of ethylene-responsivegenes in wild-type and transgenic plants. Primers were 5 0 -TTA- ARTICLE IN PRESS A novelERFgene of rice 1719 CAACGCCTTTATCACCG-3 0 and 5 0 -ACCACCAGGATTAACAC- CAA-3 0 for b-chitinase gene (At3G12500); 5 0 -TACATCT- ATACATTGAAAAC-3 0 and 5 0 -ACAACGGGAAAATAAACA-3 0 for PDF1.2 gene (At5G44420). ACTIN 2 (At3G18780) was used as a constitutive control with primers (5 0 -CTGTGCCAATC- TACGAGGGT-3 and 5 0 -GCTGGAATGTGCTGAGGGA-3 0 ). PCR involved a total of 25 cycles. Results OsERF1 is a novelERF-like gene Using the known AP2/EREBP DNA-binding domain sequence of EREBP1 as a query, we performed the tblastn search against the TIGR rice genomic database and retrieved several genomic sequences encoding putative ERFgenes. One of them, designated as OsERF1, was selected for further investigation since sequence analysis indicated that OsERF1 was a potential ethylene-responsivegene (for details, see description below). We cloned its full-length cDNA of 1589 bp long by a RACE approach (sequence data from this article have been deposited at GenBank under accession no. EF061888). Sequence analysis shows that OsERF1 has an open-reading frame (OFR) of 957 bp capable of encoding a 318 amino acids long protein. The predicted protein has a calculated molecular mass of 33.1 kD and a pI of 4.83, with a relatively hydrophilic feature. By the blastn search against the TIGR rice genomic database with the full-length cDNA, we retrieved only one hit with 100% identity and no other significantly similar ones over the cDNA sequence, suggesting that OsERF1 exists as a single copy gene. The genomic sequence of OsERF1 localizes in chromosome 4 (Os04g46220), spans nucleotides 81171–82127 of BAC clone OSJN- Ba0079A21 (accession no. AL607006), and has no intron. OsERF1 contains a single AP2/EREBP DNA-binding domain (AAs 151–210) (Figure 1A), an alanine-rich region of 111 amino acids (AAs 119–229) and a serine-rich region of 25 amino acids (AAs 241–265). Moreover, the presence of a nuclear-targeting motif (AAs 266–270) is consistent with the nucleus localization feature of the protein, which was observed experimentally (Figure 2). Multiple sequence alignment of OsERF1 and other known AP2/EREBP proteins showed their similarity restricted intensively to the AP2/EREBP DNA-binding domain region. Over this region, OsERF1 shared high sequence identity (X80.83%) with other ERFs, relatively low identity (p65.98%) with DREBs and considerably low identity (p37.7%) with APETALA2 proteins (Figure 1A). In addition, the conserved